báo cáo hóa học: " Formation of multinucleated giant cells and microglial degeneration in rats expressing a mutant Cu/Zn superoxide dismutase gene" ppt

Methods: Multiple levels of the CNS from spinal cord to cerebral cortex were studied in SOD1G93A transgenic rats during three stages of natural disease progression, including presymptoma

Open Access

Research

Formation of multinucleated giant cells and microglial

degeneration in rats expressing a mutant Cu/Zn superoxide

dismutase gene

Sarah E Fendrick, Qing-Shan Xue and Wolfgang J Streit*

Address: Department of Neuroscience, University of Florida College of Medicine and McKnight Brain Institute, 100 Newell Drive, Gainesville FL

32611, USA

Email: Sarah E Fendrick - sefendrick@yahoo.com; Qing-Shan Xue - qsxue@ufl.edu; Wolfgang J Streit* - streit@mbi.ufl.edu

* Corresponding author

Abstract

Background: Microglial neuroinflammation is thought to play a role in the pathogenesis of

amyotrophic lateral sclerosis (ALS) The purpose of this study was to provide a histopathological

evaluation of the microglial neuroinflammatory response in a rodent model of ALS, the SOD1G93A

transgenic rat

Methods: Multiple levels of the CNS from spinal cord to cerebral cortex were studied in

SOD1G93A transgenic rats during three stages of natural disease progression, including

presymptomatic, early symptomatic (onset), and late symptomatic (end stage), using immuno- and

lectin histochemical markers for microglia, such as OX-42, OX-6, and Griffonia simplicifolia isolectin

B4

Results: Our studies revealed abnormal aggregates of microglia forming in the spinal cord as early

as the presymptomatic stage During the symptomatic stages there was prominent formation of

multinucleated giant cells through fusion of microglial cells in the spinal cord, brainstem, and red

nucleus of the midbrain Other brain regions, including substantia nigra, cranial nerve nuclei,

hippocampus and cortex showed normal appearing microglia In animals during end stage disease

at 4–5 months of age virtually all microglia in the spinal cord gray matter showed extensive

fragmentation of their cytoplasm (cytorrhexis), indicative of widespread microglial degeneration

Few microglia exhibiting nuclear fragmentation (karyorrhexis) indicative of apoptosis were

identified at any stage

Conclusion: The current findings demonstrate the occurrence of severe abnormalities in

microglia, such as cell fusions and cytorrhexis, which may be the result of expression of mutant

SOD1 in these cells The microglial changes observed are different from those that accompany

normal microglial activation, and they demonstrate that aberrant activation and degeneration of

microglia is part of the pathogenesis of motor neuron disease

Published: 28 February 2007

Journal of Neuroinflammation 2007, 4:9 doi:10.1186/1742-2094-4-9

Received: 12 January 2007 Accepted: 28 February 2007 This article is available from: http://www.jneuroinflammation.com/content/4/1/9

© 2007 Fendrick et al; licensee BioMed Central Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Journal of Neuroinflammation 2007, 4:9 http://www.jneuroinflammation.com/content/4/1/9

Background

Amyotrophic lateral sclerosis (ALS) is an adult onset

neu-rodegenerative disease characterized by selective loss of

upper and lower motor neurons Loss of motor neurons

results in muscle paralysis and ultimately death due to

res-piratory failure 5–10% of ALS cases are familial inherited

in an autosomal dominant pattern, and of familial ALS

cases 20% have been linked to mutations located in the

Cu/Zn superoxide dismutase 1 (SOD1) gene [1-4] The

discovery that SOD1 gene mutations are linked to motor

neuron disease has facilitated development of transgenic

rodent models to mimic human disease [1,2,5], and these

have provided important leads towards understanding the

molecular pathology of ALS Since SOD1 is critically

involved in eliminating superoxide, an undesirable

byproduct of oxidative phosphorylation and a potential

source of oxidative damage, the fact that transgenic

ani-mals with SOD1 mutations show unchanged or even

ele-vated SOD1 activity has led to the conclusion that it is not

a lack of enzymatic activity that contributes to disease

development but rather some acquired toxic property of

the enzyme [6,7] Thus the question arises, what are the

cellular targets of this toxicity? Several studies have shown

that expression of mutant SOD1 limited to motor

neu-rons is insufficient to cause motor neuron degeneration

[8,9], and work by Cleveland and co-workers has

gener-ated findings, which show that toxicity to motor neurons

requires damage from mutant SOD1 acting within

non-neuronal cells [10] and, more specifically, that microglial

cells are important for late stage disease development

[11] These findings point towards a critical involvement

of microglia in motor neuron disease development, yet

the nature of microglial-neuronal interactions that lead to

motor neuron degeneration remains unknown One

pos-sibility, which has also been studied extensively in the

context of other neurodegenerative diseases, notably

Alzheimer's disease, is the notion of chronic and

detri-mental microglial neuroinflammation [12] According to

this theory, activated microglia are seen as the main

cellu-lar source of inflammatory mediators in the CNS and as

such are thought to be potentially neurotoxic [13,14]

Chronic neuroinflammation is thought to be involved

also in the pathogenesis of ALS based on a variety of in

vivo and in vitro studies concerned with studying

micro-glial activation using both human and animal tissues

[15-20]

In order to learn more about the role of microglia in the

pathogenesis of motor neuron disease, we set out to

inves-tigate microglial activation in the G93A SOD1 mutant rat

during natural disease progression The results reported

here are unexpected in that they reveal a highly abnormal

microglial reaction that does not meet the criteria of an

anticipated, characteristic neuroinflammatory response

Methods

Animals

Animal use protocols were approved by the University of Florida Institutional Use and Care of Animals Committee (IUCAC) All transgenic animals used in this study were male Sprague Dawley NTac:SD-TgN(SOD1G93A)L26H rats obtained from Taconic Farms where animals were screened extensively for infections prior to shipping Upon arrival animals were housed under SPF conditions Age-matched, wild type Sprague Dawley rats were pur-chased from Harlan The time course of disease progres-sion varied among individual animals, but in general once symptoms developed disease progression was quite rapid causing death of most animals by 5 months of age

To examine microglial morphology, microglial markers were used at three stages of the disease: 1) presympto-matic stage, where animals had no apparent muscle weak-ness Animals studied in this group were aged 74–84 days; 2) early symptomatic stage (onset), where animals first showed evidence of hind limb weakness Animals studied

in this group were aged 113–117 days; 3) late sympto-matic (end stage), where animals were no longer able to right themselves after 30s Animals studied in this group were aged 135–156 days For each of the three disease stages, 4 transgenic and 4 age-matched wild type control animals were used

Tissue processing and immunohistochemistry

Animals were deeply anesthetized with pentobarbital and perfused transcardially with phosphate buffer saline (PBS) followed by a fixative solution containing 4% para-formaldehyde in PBS The spinal cord and brain were dis-sected out and fixed overnight in 4% paraformaldehyde at 4°C, transferred to 30% sucrose and then frozen Lumbar spinal cord, cortical, and brainstem sections were cut in the coronal plane at 20 μm on a cryostat, mounted on slides and air dried Sections were pretreated in PBS with 0.5% Triton X-100 for 15 min, blocked in 10% normal goat serum for 30 min and incubated overnight at room temperature in the primary antibody diluted in buffer The primary antibodies included MRC OX-42 (Serotec, Cambridge, UK) and MRC OX-6 (Serotec, Cambridge, UK) at 1:500 The slides were rinsed in PBS and incubated

in secondary antibody (1:500) for 1 h Following incuba-tion, slides were rinsed and Horseradish Peroxidase Avi-din D was applied (1:500; Vector, Burlingame, CA) and incubated for 30 min Slides were washed and immunore-activity was visualized with 3,3'-diaminobenzidine (DAB)-H2O2 substrate After a brief rinse, slides were dehydrated in increasing concentrations of ethanols, cleared in xylene, and coverslipped using Permount mounting medium (Fisher Scientific)

OX-42 immunoreactivity in the ventral spinal cord was

quantified using Image Pro Plus software (version 4.5.1,

Media Cybernetics, Carlsbad, CA) The area occupied by

stained cells was highlighted and measured for each

sec-tion of spinal cord (6 secsec-tions per animal) then expressed

as a percentage of total area of ventral spinal cord Using

GraphPad Prism software (San Diego, CA) a t-test was

per-formed to determine statistical significance between

trans-genic SOD 1 and control animals at each time point A

one-way ANOVA was performed to compare differences

among the transgenic animals followed by a Tukey

multi-ple comparison test

Paraffin processing and lectin histochemistry

Animals were deeply anesthetized and transcardially

per-fused with phosphate buffer saline (PBS) followed by a

fixative solution containing 4% paraformaldehyde The

spinal cord and brain were dissected out and fixed 2 h in

4% paraformaldehyde The tissue was dehydrated

through ascending alcohols, cleared in xylenes and

embedded in paraffin Serial 7 μm coronal sections were

collected and mounted on slides Sections were

deparaffi-nized through xylenes, graded alcohols and rinsed in PBS

Next, the slides were trypsin treated (0.1% trypsin, 0.1%

CaCl2) for 12 min at 37°C Following a 10 min wash the

slides were incubated overnight at 4°C in lectin GSA I-B4

-HRP (Sigma Chemical Co.) diluted 1:10 in PBS

contain-ing cations (0.1 mM of CaCl2, MgCl2 and MnCl2) and

0.1% Triton X-100 After overnight incubation slides were

briefly rinsed in PBS and visualized with

3,3'-diabi-mobenzidine (DAB)- H2O2 substrate Sections were

coun-terstained with cresyl violet, dehydrated through

ascending alcohols, cleared in xylenes and coverslipped

with Permount

Results

Development of microgliosis during natural disease

progression in the spinal cord

The CR3 complement receptor recognized by OX-42

anti-body is expressed constitutively by all resting and

acti-vated microglial cells [21] OX-42 immunoreactivity

observed in presymptomatic SOD1 transgenic rats was

similar to that seen in wild type control, i.e there was

uni-form staining of all resting microglia (Figs 1A,D)

Occa-sionally, in these presymptomatic animals cell fusions

involving several microglia were observed (Fig 1A, inset)

The onset of symptoms was associated with a dramatic

increase in OX-42 staining in the ventral horn due to

much greater microglial cell numbers (Fig 1B) Many of

these seemingly activated microglia were clustered and/or

fused into multi-cellular aggregates In end stage animals,

overall immunoreactivity with OX-42 was decreased

com-pared to that seen in animals with disease onset (Fig 1C)

This unexpected diminution in microglial staining was

due to widespread degenerative cytoplasmic

fragmenta-tion affecting most, if not all microglia within the ventral horn (see below) The qualitatively evident increases and decreases in immunoreactivity were confirmed through quantitative morphometric measurements (Fig 1E) With onset of symptoms, there was apparent activation of microglia as judged by the dramatic increase in OX-42 immunoreactivity in the spinal gray matter Examining sections at low power clearly revealed pronounced spots

of enhanced OX-42 staining in the ventral horns (Fig 2A), and these were judged initially to be due to the formation

of microglial phagocytic clusters around dying motor neu-rons, as this would be a normal response to motor neuron death However, when spots of intense OX-42 immunore-activity were examined at higher power (Fig 2B,D) they appeared unusual in that individual microglial phago-cytes were not discernable Subsequent counterstaining of these sections with cresyl violet allowed us to conclude that the OX-42 reactive structures were, in fact, not phago-cytic clusters but represented multinucleated giant cells (Figs 2C,E) These giant cells were found in all SOD1G93A

transgenic rats studied They formed apparently as a result

of multiple microglial cells fusing together into sizable syncytia (40–50 μm) that often showed a circular arrange-ment of microglial nuclei about their periphery (Fig 2E) This kind of nuclear arrangement is classically associated with multinucleated giant cells of the Langhans type The cytoplasmic interior of Langhans giant cells appeared granular and fragmented, suggesting ongoing deteriora-tion A few of the giant cells revealed the presence of apop-totic bodies, evident as nuclear fragments (Fig 2F), but overall apoptotic bodies either inside or outside of giant cells were sparse Microglia dispersed in between giant cells revealed relatively normal process-bearing morphol-ogy and lacked the conspicuous hypertrophy that is char-acteristic of activated microglia (Fig 2E) However, some sections showed ongoing microglial cytorrhexis, i.e frag-mentation of the cytoplasm Cytorrhexis became conspic-uous in animals that were in the terminal stages of the disease process (Fig 3) and was evident as a loss of dis-cernable microglial cell structure and presence of abun-dant OX-42 immunoreactive fragments of microglial cytoplasm dispersed throughout the spinal gray matter (Figs 3A,B,D,E) Occasional giant cells could still be observed during end stage disease, however, most of these showed signs of deterioration evident by increased irregu-larity of their shape and nuclear arrangement, as well as by increased granularity and fragmentation (Figs 3A,B) In some sections, neurons remained stained with cresyl vio-let suggesting residual preservation of neuronal integrity However, pathological features were evident in motor neurons, including most notably intense hyperchromia with formation of a nuclear cap consisting of condensed chromatin material (Fig 3C) This neuronal appearance

Journal of Neuroinflammation 2007, 4:9 http://www.jneuroinflammation.com/content/4/1/9

Microglial staining with OX-42 immunohistochemistry in the spinal cord during three different stages of motor neuron disease progression

Figure 1

Microglial staining with OX-42 immunohistochemistry in the spinal cord during three different stages of motor neuron disease

progression A, presymptomatic stage; inset shows early microglial fusion in spinal cord B, disease onset; C, end stage; D, wild type control Note the dramatic increase in microglial staining with OX-42 during onset (B) and its subsequent decline during end stage (C) Scale bar: 200 μm E, morphometric quantification of microglial immunostaining with OX-42 during disease

development; * p < 0.05 and ** p < 0.001 with respect to age-matched controls; # p < 0.05 with respect to onset group

stood in stark contrast to that of normal motor neurons as

seen in wild type animals (Fig 3F)

Histopathology in the brain stem

Sections from the brainstem at the level of cranial nerve

VII during disease onset and end stage were marked by

changes indicative of severe neuropathology (Fig 4) They

included prominent, widespread vacuolization of the

extracellular space and hyperchromia of neuronal

proc-esses Often neurites appeared physically separated (as if

torn) from neuronal cell bodies leaving one or more

dis-tinct stumps on the perikaryon (Fig 4D,E) The changes

affecting microglia were striking in that multinucleated

giant cells were present throughout any given section

These consisted of fused microglial cells that gave rise to a

variety of bizarrely shaped cellular fusions which, in some

cases, extended for more than one hundred micrometers

in length (Figs 4B,C,F) Microglial fusions varied in size,

sometimes involving only a few cells, and other times

twenty or more Although not obviously associated with

vascular channels, some microglial giant cells due to their

elongated shape seemed to have formed along blood

ves-sels (Fig 4C) Presence of giant cells was observed in all

animals regardless of whether they were at an early or late

symptomatic stage of motor neuron disease They were

scattered seemingly at random throughout the brainstem

and not limited to any particular nucleus or tract, and

often displayed the classic morphological features of

Langhans type giant cells (Fig 4G)

Within vacuolated spaces rounded, shrunken microglia

exhibiting nuclear fragmentation or shrinkage (pyknosis)

were identified using lectin histochemical staining (Figs

4H)

Microglia in midbrain and cerebral cortex

Microglial fusions similar to those seen in the spinal cord

and brainstem level were found also in the red nucleus of

the midbrain (Figs 5A–D) The specificity with which

these microglial fusions were restricted to the red nucleus

area was remarkable, as they were visible even at the

low-est magnification (Fig 5A) Microglia outside of the red

nucleus displayed normal, ramified morphology

Rubros-pinal neurons appeared normal in size and morphology,

as well as in number, and there was no evidence to suggest

that any of these neurons were undergoing degeneration

Rubrospinal neurons were not encircled by activated

microglia It is noteworthy also that motor neurons in the

oculomotor nucleus, which appears with the red nucleus

in the same sections, revealed no evidence of degenerative

changes, and microglia here were normal and

non-acti-vated in appearance Similarly, microglia in the substantia

nigra appeared completely normal (Fig 5F) Somewhat

surprisingly, we also found no evidence at all for

micro-glial activation or abnormalities in the motor cortex of

animals, regardless of disease stage, with any of the micro-glial markers employed (Figs 5G,H)

Discussion

The purpose of the current study was to perform an inves-tigation of microgliosis in a recently developed rat model

of ALS involving expression of a mutated human SOD1 transgene (G93A) [5] Although these animals, similar to their murine counterparts, reportedly mimic many of the histopathological features of human ALS, including glial activation [5,19], until now a detailed analysis of reactive microgliosis has not been performed Our current results show that the microgliosis that occurs in SOD1G93A rats is atypical and marked by some highly unusual features in microglial cells that are indicative of cellular dysfunction The key microglial aberrations found consist of fusion into giant cells and cytorrhexis (Fig 6) These features are not observed normally during microglial activation and they lead us to conclude that this particular animal model

of ALS is characterized by microglial degeneration rather than by microglial neuroinflammation It is therefore con-ceivable that neurodegeneration occurs as a consequence

of glial cell deterioration

Prior work in ALS rodent models involving SOD1 muta-tions has generated clues about an involvement of glial cells Damage to astrocytes has been described to occur concomitant with degeneration of motor neurons prompting the hypothesis that astrocytic damage pro-motes motor neuron degeneration [22] However, subse-quent experiments showed that restricted expression of mutant SOD1 genes in astrocytes is not sufficient to cause motor neuron degeneration [23] Notwithstanding these findings, more recently it was determined using chimeric animals consisting of mixtures of normal cells and cell expressing human mutant SOD1 that nonneuronal cells containing mutant SOD1 are indeed required to cause damage to motor neurons, whereas wildtype nonneuro-nal cells promote motor neuron survival [10,24] In addi-tion, recent work has shown that mutant SOD1 acting within microglial cells specifically is a primary determi-nant of late stage disease progression [11] These observa-tions gain added significance when considered together with the current findings showing widespread microglial degeneration in the spinal cord gray matter of end stage animals, because it now seems clear that mutant SOD1 is particularly toxic to microglia and that SOD1-mediated microglial degeneration is linked to a terminal neurode-generative disease state Thus, loss of microglial cells could be very detrimental to neuronal survival [25] Future research may be directed towards elucidating the molecular mechanisms that underlie SOD1's selective microglial toxicity, and towards ways of inhibiting it as a strategy for new ALS treatments

Journal of Neuroinflammation 2007, 4:9 http://www.jneuroinflammation.com/content/4/1/9

OX-42 immunohistochemistry during symptomatic phase of disease

Figure 2

OX-42 immunohistochemistry during symptomatic phase of disease A, low power view reveals intensified immunoreactivity in spinal cord ventral horns; multiple large, rounded spots are visible B, higher power view of large immunoreactive spots is sug-gestive of phagocytic clusters C, same field as in B; counterstaining with cresyl violet facilitates identification of large immuno-reactive spots as multinucleated giant cells D, E, the same microscopic field prior to and after cresyl violet counterstaining reveals a well-formed multinucleated giant cell of the Langhans type F, enlargement of framed area in C shows apoptotic

microglial nucleus (arrow) within a giant cell Scale bars: 500 μm (A), 40 μm (B, C), 20 μm (D-F)

OX-42 immunohistochemistry during end stage disease demonstrates extensive microglial cytoplasmic fragmentation (A-E)

Figure 3

OX-42 immunohistochemistry during end stage disease demonstrates extensive microglial cytoplasmic fragmentation (A-E)

A, D, two different views of spinal ventral gray matter demonstrate loss of microglial cell integrity and widespread punctate staining indicative of cytorrhexis Note that many neurons remain stained with cresyl violet B, enlargement of framed area in

A shows detail of microglial cytorrhexis, including a disintegrating giant cell on the right E, enlargement of framed area in D shows detail of microglial cytorrhexis C, motor neuron in SOD1G93A rat reveals intense hyperchromasia with cresyl violet and

nuclear cap F, normal motor neuron and microglia from wild type spinal cord Scale bars: 40 μm (A, D); 20 μm (B, C, E, F)

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Lectin staining of microglia in the brainstem (level of cranial nerve VII) in wildtype animals (A) and in late symptomatic/end stage animals (B-H)

Figure 4

Lectin staining of microglia in the brainstem (level of cranial nerve VII) in wildtype animals (A) and in late symptomatic/end stage animals (B-H) Cresyl violet counterstain A, microglia show normal ramified morphology B, a large lectin-positive

aggregate of fused microglia is evident in severely vacuolated brainstem tissue Note enlarged perineuronal spaces to the right

C, string-like microglial fusions extend over long distances D, breakage of neuronal process, probably a dendrite, from cell body within markedly vacuolated space (arrows) E, two multinucleated microglial giant cells are seen below a neuron with broken off process (arrow) F, large multinucleated giant cell displaying vacuolization is present amidst numerous microglial cytoplasmic fragments G, multinucleated giant cell of the Langhans type displaying characteristic peripheral arrangement of nuclei H, rounded lectin-positive microglial cell (arrow) within vacuolated space displays nuclear fragmentation indicative of

apoptosis Scale bars: 20 μm (A-H)

Visualization of microglia in midbrain with GSA-I-B4 lectin (A-F) and in motor cortex with OX-42 (G) and OX-6 (H) during

symptomatic disease

Figure 5

Visualization of microglia in midbrain with GSA-I-B4 lectin (A-F) and in motor cortex with OX-42 (G) and OX-6 (H) during symptomatic disease A, low power view of midbrain reveals enhanced lectin staining in the red nucleus B, higher

magnifica-tion shows that enhanced lectin reactivity is confined strictly to red nucleus region (arrows indicate perimeter of red nucleus)

C, microglial fusions are interspersed with rubrospinal neurons that appear undamaged D, lectin-positive microglial fusion (giant cell) within red nucleus E, oculomotor nucleus reveals normal-appearing motor neurons and lack of microgliosis F, sub-stantia nigra (pars compacta) shows presence of normal, ramified microglial cells G, motor cortex shows normal, ramified microglia H, single, ramified microglial cell positive with OX-6 (arrow) near lateral ventricle Scale bars: 400 μm (A); 200 μm (E); 100 μm (B,H); 50 μm (C,F,G); 20 μm (D)

Journal of Neuroinflammation 2007, 4:9 http://www.jneuroinflammation.com/content/4/1/9

We use the term "cytorrhexis" to describe the kind of

microglial degeneration we observed in SOD1G93A rats

because it involves disintegration of the cell's cytoplasm

rather than of its nucleus Cytorrhexis has been used

pre-viously only to describe neuronal necrosis resulting from

excitotoxicity [26], but extending its use to describe

micro-glial cytoplasmic deterioration is appropriate since this

form of cell death does not involve the nuclear

disintegra-tion (karyorrhexis) that is characteristic of apoptosis

Cyt-orrhexis therefore describes accidental, rather than

programmed, microglial cell death Our inability to detect

large numbers of apoptotic microglia in the tissues

stud-ied indirectly supports the idea that cytorrhexis is the

"pre-ferred" mode of microglial cell death during the toxic

disease state thought to be generated by mutant SOD1

expression Finding widespread microglial degeneration

in this particular animal model of neurodegenerative

dis-ease strongly supports the broader concept that microglial

abnormalities characterize other neurodegenerative

con-ditions as well [27-29]

Perhaps the earliest sign of an aberrant microglial

response in SOD1 mutant rats is reflected in our

observa-tion of occasional microglial fusions in presymptomatic

animals We suspect that with disease onset these progress

to produce the conspicuous multinucleated giant cells

The occurrence of microglial giant cells throughout the lumbar spinal gray matter, as well as the brainstem, and especially their selective localization in the red nucleus, raises the intriguing possibility that their formation is related to the fact that these regions all give rise to fibers that project onto ventral motor neurons It is conceivable therefore that a signal is transmitted retrogradely from ventral horn cells to these supraspinal regions to trigger formation of microglial fusions, consistent with the notion of disease spread from an initially affected region [11] However, at the same time the notable absence of microglial abnormalities and/or activation in the motor cortex reported here would argue against this idea Addi-tional studies providing more detailed mapping of the location of giant cells could be helpful in this regard Fusion of microglia into giant cells represents an anoma-lous type of cellular behavior, since microglia are nor-mally "territorial" and exhibit strong contact inhibition Microglial giant cells have never been described to occur

in situ in rat brain, but they can form spontaneously in vitro using cultured microglia from a variety of species

[30-33] Multinucleated giant cells are a pathological hallmark

in human brain during infectious diseases, most notably

in HIV/AIDS encephalopathy [34,35], and since microglia are the main cellular target of HIV-1 in the brain it is

Schematic depicting the approximate time course of motor neuron disease development and the accompanying microglial changes in SOD1G93A rats

Figure 6

Schematic depicting the approximate time course of motor neuron disease development and the accompanying microglial changes in SOD1G93A rats Note that disease onset and subsequent development of end stage disease is variable among individ-ual animals